Microbial production of polyfructose

ABSTRACT

A water-soluble levan having a weight average molecular weight of about 10,0000-40 million, preferably about 5-25 million and especially about 10-20 million which stabilizes a bovine serum albumin colloid having index (EAI) of about 3-100 determined according to the formula EAI=2T/OC wherein T is turbidity measured at 500 nm, C is the weight of emulsified protein per unit volume of the aqueous phase, and O is the volume fraction of a dispersed oil phase is produced by fermenting a nutrient growth medium feedstock having a carbon source consisting essentially of an assimilable sugar selected from the group consisting of sucrose, raffinose, or a mixture thereof with a Zymomonas mobilis microorganism capable of converting at least 50% of the fructose value of the sugar to said levan under nutrient growth conditions which enhance levan production while suppressing ethanol production. It is useful as a colloid stabilizing agent, particularly with foods, beverages pharmaceuticals, dentrifices, and cosmetics.

DESCRIPTION OF THE INVENTION Technical Field of the Invention

This invention relates to a water-soluble, essentially uncharged levanpolymer which has useful emulsifying and thickening propertiescomparable to those of gum arabic, and to methods for the production anduse thereof as well as compositions containing said levan polymer as anemulsifying agent. More particularly, this invention relates to theproduction of levan through the action of levan sucrase produced by animproved and modified bacterial strain of Zymomonas mobilis.

BACKGROUND ART

Hydrocolloids, particularly polysaccharide gums, are widely employed inthe food industry to stabilize disperse foods such as emulsions andfoams (e.g. ice cream, whipped toppings, beer, salad dressings,sausages, etc.) see S. C. Sharma, Gums and Hydrocolloids in Oil WaterEmulsions, J. Food Tech. January 1981, 59-67. These foods arepolydisperse colloidal systems and are generally thermodynamicallyinherently unstable, i.e. in the absence of specific stabilizing agentssuch as polysaccharides the physical state of the processed food rapidlydeteriorates. For example, ice creams develop ice crystals and becomegrainy, salad dressings separate, and sausages both exude oil andexhibit syneresis or weeping. The precise role of the polysaccharide isnot well established in complex foods in which they are used; however,their ability in model systems to modify both the bulk properties of thecontinuous aqueous phase and the interfacial behavior of othercomponents, particularly proteins, is considered indicative of theirbeneficial effects.

Levan or anhydrofructosylfructoside is a linear polymer of adjacentfructose residues covalently bonded together in the beta conformationbetween the 2- and 6- carbon atoms (E. A. Dawes et al., Biochem. J. 98:804-812, 1966.) This polymer can be formed by the microbial fermentationof sucrose through a transfructosylation reaction catalyzed by theenzyme levan sucrase (E.C. 2.4.1.10). Several strains of bacteria havebeen described which elaborate a functional levan sucrase. These includestrains of Actinomyces viscosus, Aerobacter levanicum, Acetobactersuboxydans, Bacillus licheniformis, Bacillus macerans, Bacillussubtilus, Gluconobacter oxydans, Streptococcus salivarius, Streptococcusmutans, Leuconostoc mesenteroides, Erwinia herbicola and Zymomonasmobilis. Levan can also be formed in vitro by the action of levansucrase on either sucrose or raffinose which functions as a fructosyldonor and an aldose molecule which acts as the fructosyl acceptor.

Levan has been used as an immunologic suppressor (Experientia 34:1362-1363, 1978 and J. Pathol. 125: 103, 1978) and an immunogenic haptenconjugate (J. Dent. Res. 55: 129, 1976 and Moreno, U.S. Patent No.4,260,602). UK Patent Application GB No. 2,046,757-A published Nov. 19,1980 describes a low cariogenic food sweetener prepared by subjecting asubstrate solution containing aldose and sucrose or raffinose to theactivity of levan sucrase, but the desired product is a trisaccharideand not a high molecular weight levan, which has not heretofore been toprovide functional properties to food products.

Traditionally, small quantities of levan have been produced by thebacterial fermentation of sucrose, usually by a strain of Actinomycesviscosus or Aerobacter levanicum. Levan produced by the in vitroactivity of levan sucrase has been reported by T. Tanaka et al. in J.Biochem. 87 (1): 297-303 (1980). The applications of recombinant DNAtechnology have been applied in the vitro genetic transfer of the sacBgene from Bacillus subtilis into a strain of Escherichia coli (J.Bacteriol. 153: 1424-31, 1983.) These processes, however, do notrepresent efficient means for the production of levan. In particular,the in vitro enzymatic synthesis of levan from sucrose would appear tobe difficult in terms of producing a high molecular weight molecule, asevidenced by UK Patent Application GB No. 2,046,757-A.

The in vivo production of levan has also been described, e.g. in Jeaneset al. U.S. Pat. No. 2,673,828; Gaffar et al. U.S. Pat. No. 3,879,545;and Ayerbe et al. U.S. Pat. No. 4,399,221, the contents of which areincorporated by reference herein. Such methods have generally employedaerobic fermentation techniques in the presence of relatively smallamounts of sucrose (i.e., less than about 2%) in the feedstock and haveachieved relatively low yields.

DISCLOSURE OF THE INVENTION

Accordingly, it is a general object of the present invention to providecolloid systems which are stabilized by an effective amount of awater-soluble levan having a molecular weight of about 10,000-40million, preferably about 5-25 million and especially about 10-20million and which characteristically stabilizes a bovine serum albuminemulsion having an emulsion activity index (EAI) of about 3-100,preferably about 10-50 and especially of at least 20, determinedaccording to the formula EAI=2T/OC wherein T is turbidity measured at500 nm, C is the weight of emulsified protein per unit volume of theaqueous phase, and O is the volume fraction of a dispersed oil phase.

Another object of the present invention is to provide such stabilizedcolloidal systems in the form of an emulsion, aerosol, or foam whereinthe colloidal components are admixed with an effective amount of a levanstabilizing agent according to the present invention.

An additional object of the present invention is to provide a processfor forming a stable emulsion of a water-insoluble or water-immisciblecompound in water by admixing said compound with water, an emulsifyingagent, and a stabilizing amount of a levan according to the presentinvention to provide emulsions having improved stability.

Yet another object of the present invention is to provide a process forforming a stable colloidal aerosol of a liquid dispersed in a gas byadmixing said components with a suitable colloid forming agent and astabilizing amount of a levan according to the present invention toprovide aerosols having improved stability.

A further object of the present invention is to provide a process forforming a stable colloidal foam of a gas dispersed in a liquid byadmixing said components with a foaming agent and a stabilizing amountof a levan according to the present invention to provide foams havingimproved stability.

A more particular object of the present invention is to provide stableemulsions of foods and beverages, particularly protein emulsions such asprotein-air colloids or whipped protein food products.

Still another object of the present invention is to provide abiologically pure, stable in vitro culture of a microorganism from thegenus Zymomonas which has been adapted to ferment sucrose or raffinosein a fermentation culture medium containing assimilable sources ofcarbon and nitrogen to form a levan product which is useful according tothe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will become more fully apparent to those skilled in the art towhich this invention pertains from the following detailed description,taken in conjunction with the annexed drawings, wherein:

FIG. 1 plots the drainage of whipped egg white foams against whippingtime and drainage time to illustrate the sensitivity of the method usedherein for determining emulsion activity and stability;

FIG. 2 shows the effect of levan on the emulsion activity index (EAI) ofbovine serum albumin (BSA);

FIG. 3 illustrates the effect of levan on the emulsion stability ofbovine serum albumin; and

FIG. 4 illustrates representative drainage rates of foams prepared withBSA and levan according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Briefly, the above and other objects, features and advantages of thepresent invention are attained in one aspect thereof by providing newtechniques for the preparation and use of a water-soluble levan having aweight average molecular weight of about 10,000-40 million, preferablyabout 5-25 million and especially about 10-20 million and which isfurther characterized by stabilizing colloids such as bovine serumalbumin oil emulsions having an emulsion activity index (EAI) of about3-100, preferably about 10-50 and especially of at least 20, determinedaccording to the formula EAI=2T/OC wherein T is turbidity measured at500 nm, C is the weight of emulsified protein per unit volume of theaqueous phase, and O is the volume fraction of a dispersed oil phase.While higher EAI values of up to about 200 can be obtained, they areundesirable because the emulsion stability is generally reduced tounacceptably low levels.

While described in detail with respect to the presently preferredembodiment of a colloidal foam system of air in protein such as eggwhite or BSA, it will be appreciated by those skilled in the art towhich this invention pertains that the levan of the present inventioncan be used to stabilize diverse types of colloidal systems includingsols (Dispersions of solid in liquid), emulsions (dispersions of liquidsin liquids), aerosols (dispersions of liquids or solids in gases), foams(dispersions of gases in liquids or solids), and gels (dispersions inwhich one component provides a structual framework with other componentsfilling the interstitial spaces therein).

In one aspect, the present invention provides a new method for thepreparation of such levans. In accordance with this aspect, alevan-producing microorganism is acclimated to grow anaerobically on aculture medium having as the sole source of assimilable carbon atoms oneor more sources of the sugars sucrose, raffinose, or mixtures thereof atincreasingly high concentrations of from at least about 2% up to about40% of such sugar, generally at least 5% and preferably about 10-15%.The culture medium contains a suitable source of assimilable nitrogenatoms, but in amounts less than half that normally required when beinggrown for ethanol production, preferably less than one-fourth of suchamount. Vitamin requirements such as pantothenic acid, biotin, thiamine,pyridoxine, and nicotinic acid can be provided as yeast extractcomponents or separately. Additionally, fermentation is carried out attemperatures below those employed in ethanol production, generally atleast 5° C. and preferably at least 10° C. lower, thereby furtherfavoring the microorganism's metabolism in favor of producing levan fromsucrose.

Optimal results to date have been achieved within the above parametersfor Zymomonas mobilis by first passing the inoculum seed culture 3-4times in a culture medium containing sucrose as the sole carbon sourceat concentrations of preferably 10-15%. A suitable yeast extract isemployed as the sole nitrogen source at concentrations of only about0.25%, since in some instances levan production has been observed todrop at only 0.5% (yeast extract is generally used at concentrations of1-2% for ethanol production). Fermentation at room temperature of about25° C. compared to about 30° C. for ethanol production has provenadvantageous.

Especially preferred is the use of such a culture technique with amicroorganism which has been selectively adapted and isolated for thepreferential production of levan under such conditions, e.g. by repeatedsubculture and selection of a levan-producing microorganism inincreasing concentrations of sucrose under the above conditions.Presently preferred for such applications is a strain of Zymomonasmobilis which has been described by E. L. Dally in "Plasmid Studies inZymomonas mobilis", Master's Thesis, Rutgers, The State University ofNew Jersey, New Brunswick, N.J. (1982), particularly a variant thereofwhich has been deposited with the American Type Culture Collection,Rockville, Md. 20850 as ATCC strain No. 39,985.

The microorganism is cultivated under otherwise conventional nutrientgrowth conditions which may be modified to enhance the formation oflevan by techniques known to those skilled in the art, e.g as describedin European Patent Application Publication No. 0112661-A2 published July4, 1984. Additionally, the microbial cells can be in suspension orimmobilized onto a water-insoluble support, e.g. as described in Kim etal. U.S. Pat. No. 4,384,044. After about 72 hours, fermentation can beterminated by removal or sterilization of the microorganisms and thelevan recovered by cold ethanol precipitation, after which excessmoisture is removed by drying, preferably to a moisture content of lessthan 10% by weight, and especially to less than about 2% by weight, e.g.by freeze-drying.

The effect of levan colloidal systems has been evaluated in conjunctionwith a standard protein in model disperse systems to determine if thepolysaccharide was effective at improving the initial formation and,more importantly, the kinetic stability of dispersed emulsions and foamsformed using standard proteins.

The values obtained for EAI and colloid stability are criticallydependent on the measuring system employed. For all values used herein,foam formation and stability were monitored using a whipping methoddeveloped for the USDA Northeast Regional Project on ProteinFunctionality. 50 ml solutions of 5% BSA protein solution (0.1Mphosphate pH 7.0), with or without various concentrations of the addedlevan, were whipped for 10 minutes at speed 13 in a Sunbeam I-125 mixer.Foam stability was measured by the weight of liquid drained from thefoam and monitored continuously using an electrobalance and Applecomputer. The sensitivity of the method is illustrated by thecharacterization of the overwhelming of egg white proteins (FIG. 1).

Results to date on the effects of the levan polysaccharide on modelledcolloidal food systems suggest that this molecule, at very low levels,is able to significantly modify the physical properties of the system.Both emulsion and foam stability were improved by the presence of lessthan 0.05% levan in the aqueous phase. Evidence to date also suggeststhat these effects are not due solely to a nonspecific increase in bulkaqueous viscosity; rather, the levan appears to interact with theprotein component and significantly improve its interfacial properties.These preliminary results have not described the precise mechanism bywhich the dramatic alterations in colloidal stability are developed,which are at present not fully known. Subsequent analyses on the modelsystems and the associations formed between the levan and othercomponents present can be used to identify the basis for the physicalalterations and readily suggest other food applications for thiscompound.

Based of the above properties, it will be appreciated that the levanproduced according to the present invention has properties which renderit useful in a wide variety of emulsification applications which arelimited essentially only by the molecular weight, lack of charge, anddegree of branching in the levan molecule. The molecular weight can betailored to a desired range by controlling fermentation parameters (e.g.time, temperature and ionic strength; see T. Tanaka et al., J. Biochem.85 (1): 287-293, 1079) and by the choice of a microorganism employed,while the uncharged nature of the molecule is an inherent property whichcan, if desired, be modified by forming salts, e.g. as described inHeinz et al. U.S. Pat. Nos. 3,413,284 and 3,524,847 and Horst U.S. Pat.No. 3,380,999. These properties would appear to prevent its use incompositions such as drilling muds or the like which would be subject tohigh shear forces and heat, tending to degrade the molecule. The levanmolecules produced in accordance with the present invention areessentially linear but can contain varying degrees of branching,generally less than 20 % and more usually less than about 10%; thisproperty has not yet been fully explored.

It will nonetheless be apparent that the levan polymers have wideindustrial application as emulsifying and emulsion-stabilizing agents,particularly in foods, beverages, pharmaceuticals, dentifrices, andcosmetics. They are not sweet and have essentially no taste, and byvirtue of their high molecular weight will not pass into the bloodstreamwithout first being degraded in the digestive tract. They areparticularly valuable in stabilizing protein emulsions, especially milkand other diary products such as ice cream, sherbert, yoghurt, custards,ice lollie, cheese and cheese products, etc., and especially in whippedemulsions such as dessert toppings, salad dressings and the like, aswell as beverages such as milk shakes, orange drinks, etc. They can beused to enhance the stability of gelatinized animal food productsanalagous to K. Buckley et al. U.S. Pat. No. 4,143,171 and of meat andvegetable food products analogous to J.R. Mitchell et al. U.S. Pat. No.4,143,172. They can be used to stabilize fruit and vegetable products,e.g. formed fruit structures such as "Fruit Roll-Ups", souffles, etc. Ingeneral, the levans produced according to the present invention can beapplied in those industrial applications known for the widely usednatural product gum arabic, as reviewed by J. Adrian et al, "Chemicaland Physiological Properties of Thickening and Gelling Additives" RevueFrancaise de Dietetique 23 (88): 3-25 (1979 ) and the references citedtherein, including electrocoating and other applications in theelectronics and photography industry, e.g. see Crandall et al., U.S. PatNo. 3,137,575.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever. In the followingExamples, the temperatures are set forth uncorrected in degrees Celsius;unless otherwise indicated, all parts and percentages are by weight.

EXAMPLE 1 Preparation of Microorganism

The application described herein are based upon the use of a moleculewith a weight average molecular weight of 10 to 20 million, asdetermined by gel filtration chromatography using Bio-Rad "Biogel"agarose having a molecular weight exclusion limit of 15 million usingbacterial cells as void markers. It would be difficult to produce such alarge molecular weight species through column chromatography of sucroseby an immobilized levan sucrase enzyme. The traditional bacterialfermentation of sucrose by strains of Aerobacter levanicum results inonly 1.3 to 1.5 grams of levan per 100 milliliters of fermentation brothof the final levan product (Methods in Enzymology 8: 161-165, 1966).Production of levan by the bacterial fermentation of 2% sucrose bywildtype (or genetically unaltered) strains of Zymomonas results in only0.2 to 0.3 grams of levan product per 100 milliliters of fermentationbroth (Biochem. J. 98: 804-812, 1966). This represents the utilizationof only 2% of the total sucrose substrate present. However, from Table1, one can see that an efficient wildtype strain in 15% sucrose produces2.4 grams/100 ml compared with genetically modified and selected progenywhich produce 3.4, 2.9 and 2.9 grams/ 100 ml respectively.

                  TABLE 1                                                         ______________________________________                                                                  % Available Fructose                                Strain  Levan produced (g/100 ml)                                                                       Converted to Levan                                  ______________________________________                                        GRC 4-15                                                                              2.4               51.4                                                GRC 4-57                                                                              3.4               72.1                                                GRC 4-58                                                                              2.9               60.1                                                GRC 4-59                                                                              2.9               60.1                                                ______________________________________                                    

Strain GRC 4-57 has been serially passaged in 15% sucrose and Difcoyeast extract to allow additional adaptation to high concentrations ofsucrose. A progeny strain has been isolated from strain GRC 4-57 whichhas resulted in the production of 4.0 to 5.0 grams of levan/100 ml offermentation broth. This strain, GRC 4-57A, is capable of production oflevan at levels not previously reported in the scientific literature.This strain has been deposited with the American Type CultureCollection, Rockville, Md. 20850 U.S.A. and bears the designationZymomonas mobilis ATCC 39,985.

EXAMPLE 2 Preparation of Levan Polymer

Using a 25 liter fermentation tank containing an aqueous medium of 15%table sugar and 0.25% Amber ex 510 yeast extract, Zymomonas mobilis ATCC39,985 was inoculated (10% by volume) at room temperature underagitation (tip speed 130 feet/minute) without any extraneous pH control.After a seed time of about 48 hours, fermentation was continued forabout 72 hours, giving a yield of about 35 g/l in the broth.

Because of its intended use in food applications, the harvested brothwas continuously fed through a Sharples T-1 centrifuge at about 23,000rpm for clarification and removal of cellular debris. The clarifiedbroth was then mixed in a 1:3 ratio with ethanol and allowed to set in acold room at about 4° C. for 36 hours, during which time the levanproduct precipitated and formed a gelatinous mass on the sides andbottom of the container. After recovering the ethanol for subsequentusage, the recovered levan was freeze-dried in a lyophilizer and thedried product (pseudo-crystal plates) was then ground through a mill tothe desired particle size, e.g. of 0.1-3 mm average diameter.

The levan was readily soluble in water, forming a gel at a concentrationof about 33%. The polysaccharide was found to be difficult to completelysolubilize in buffer at various pH's; however, it readily formed aslightly turbid suspension up to high concentrations. This suspensionwas used at the specified concentrations for all subsequent analyses.

EXAMPLE 3 Emulsification Activity and Stability

The emulsification activity indices for BSA, the levan, and combinationsof each are shown in FIG. 2. The EAI estimates the total surface area ofemulsion formed using a given agitation. Bovine serum albumin isgenerally considered to produce an intermediate level of emulsion. BSAsolutions alone at 0.5% concentration formed an emulsion yielding ameans surface area of 30.3 square meters/gm. Levan by itself was aconspicuously poor emulsifier, yielding total surface areas less than 10square meters/gm at 1% concentration. The addition of the levan tosolutions of BSA did not appear to significantly affect the emulsifyingability of the protein under these conditions, i.e., the levan neitherpromoted nor prevented the BSA solution from forming an emulsion.

Emulsification activity and stability were measured using the techniquedescribed by Pearce, K. N. and Kinsella, J. E., Emulsifying Propertiesof Proteins: Evaluation of a Tubridimeteric Technique, J. Agric. FoodChem., 26(3): 716-23, 1978. The standard protein solution consisted of0.5% bovine serum albumen (BSA, fraction V, Sigma) in 0.1M phosphatebuffer pH 7.0. A measured amount of pure peanut oil and aqueous proteinand/or polysaccharide solution (10 and 30 ml respectively) werehomogenized using a blender maintained at 25° C.. for 10 seconds.Turbidity was measured at 500 nm using a Spectronic 700spectrophotometer. The emulsion activity index (EAI) was calculatedusing the formula EAI=2T/OC where T is the turbidity, C is the weight ofprotein per unit volume of the aqueous phase and O the volume fractionof dispersed oil phase. Emulsion stability was determined from thedecrease in absorbance with time.

EXAMPLE 4 Kinetic Stability of Emulsions

The effect of the levan on the kinetic stability of these emulsions isshown in FIG. 3. The presence of the levan had a profound effect on theaging of the emulsion. Whereas 60 minutes standing time resulted in a25% decrease in the total surface area of the pure BSA emulsions, overthis same time period the emulsions prepared with as little as 0.05% ofthe levan were found to actually increase in absorbance. While thisincrease is not likely to be due to an actual increase in surface areaof the emulsions, but rather an alteration in the colloidal state of thepolysaccharide, these data imply a significant improvement in thestability of the emulsion. This improvement is not solely due to anincrease in the viscosity of the continuous aqueous phase, since theeffect is not dramatically concentration dependent for thoseconcentrations tested.

The effect of the addition of levan to BSA on the foam stability wasalso dramatic, as shown in FIG. 4. The addition of as little as 0.025%polysaccharide exerted a significant improvement in the drainagestability of the foams. This was clearly not solely related to thealteration of the continuous phase viscosity for the same reason thatthe effect was not strongly concentration dependent. This suggests thatthe levan is able to alter the surface activity of the protein solutionin such a way as to improve the overall stability of foams prepared fromthem. Since the process of drainage is a highly complex interaction ofseveral rheological and colloidal parameters, significantly moreinformation would be required to establish the precise mechanism bywhich the levan is exerting the beneficial effects.

EXAMPLE 5 Foam Activity

Bovine serum albumin is also an intermediate activity foaming protein.While 5% solutions of BSA will readily form a foam, this is generallyquite unstable and collapses within 30 minutes. BSA is thus an excellentmodel protein to study the effects of protein and solution modificationson foaming properties. The levan had little foaming activity of itselfand in fact was unable to generate a measurable overrun after 20 minutesof whipping. However, addition of the levan had a significant effect onthe foams produced from 5% BSA solutions. Although the overrun was notsignificantly altered, the appearance of the foams was quite different,with levan-containing foams noticeably whiter, drier and of a finertexture. This suggested a smaller bubble size distribution in thosefoams produced from BSA in the presence of the levan.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those specifically used inthe examples. From the foregoing description, one skilled in the art towhich this invention pertains can easily ascertain the essentialcharacteristics thereof and, without departing from the spirit and scopeof the present invention, can make various changes and modifications toadapt it to various usages and conditions.

INDUSTRIAL APPLICABILITY

As can be seen from the present specification and examples, the presentinvention is industrially useful in providing new and improvedemulsifying agents which exhibit improved emulsification andstabilization activity in a wide variety of applications such as foods,beverages, pharmaceuticals, dentifrices, and cosmetics. As illustratedby the examples, the BSA acts to emulsify the oil and water phases whichare then stabilized by the Levan.

What is claimed is:
 1. In a process for the production of awater-soluble levan by anaerobically fermenting a nutrient growth mediumfeedstock having a carbon source consisting esesntially of a source ofan assimilable sugar selected from the group consisting of sucrose,raffinose, and mixtures thereof with a microorganism capable ofconverting the sugar to levan and ethanol under nutient growthconditions, the improvement wherein the microorganism is a member of thegenus Zymomonas which has the levan-producing characteristics ofZymomonas mobilis ATCC Deposit No. 39,985, the growth medium has initialsugar concentration of at least about 10% by weight and an initialconcentration of an assimilable source of nitrogen no more than abouthalf of that which would be optimal for the production of ethanol andwherein the fermentation is conducted at a temperature at least about 5°C. below the optimum temperature for the production of ethanol, therebyincreasing the amount of available fructose converted to levan.